T1DM results from the immune-mediated destruction of the body's only insulin producing cells, the pancreatic beta cells. Beta cells comprise an estimated 2% of the pancreatic cell number and are grouped together into cell clusters with alpha cells (making glucagon), delta cells (making somatostatin), F cells (making pancreatic polypeptide), and other rare cells into "mini-organs" called the islets of Langerhans. Isolated destruction of islet alpha, delta, or F cells has not been described. Isolated immune-mediated beta cell destruction does occur such that pancreata from T1DM animal models or from patients with newly diagnosed T1DM reveal "islet remnants", i.e. small islets with few or no beta cells while the other islet cell types persist. Over time however, clinical evidence suggests that patients with T1DM also lose alpha cell function suggesting that alpha cells are dependent upon normal beta cell function. The "mini-organ" concept is apt for at least 2 other reasons: (1) the different islet cell types have been thought to be organized in a typical pattern: beta cells centrally located and the other cells types located more peripherally, and (2) islets consume more pancreatic blood flow (about 20%) than their small mass would suggest. Thus islets are important, incompletely understood structures with unique physiological and immunological features, and they represent an Achilles' heel for individuals destined to develop T1DM. This project has multiple components, to: (1) improve isolation techniques to more predictably yield high quality islets, (2) improve assays for characterizing isolated islet quality, (3) test, using a non-human primate islet transplant model, important pre-clinical questions, (4) perform human clinical islet transplants using carefully planned protocols, (5) develop assays for characterizing islet function pre- and post-transplant, (6) develop a renewable islet source, and (7) test novel ways of preventing islet allograft rejection following transplant. Beginning in 7/99, in collaboration with the Clinical Center's Department of Transfusion Medicine/Cell Processing Unit and Dr. Ricordi (University of Miami's Diabetes Research Institute), islets have been isolated from >85 human pancreata and since 9/00, from >30 non-human primates. Many of these islets have been shared with various intra- and extra-mural collaborators. Before initiating the isolations, we established two separate laboratories, one for human glands and one for animals, and we trained a team of technicians. State of the art islet isolation units typically achieve islet yields approximating half the pancreatic total, and even that level is achieved only about half the time. The NIH team continues to test ways to improve islet yields. Currently available assays testing islet viability and function in vitro do not correlate with the imperfect but gold standard assay for in vivo function, i.e. islets transplanted into diabetic NOD-scid mice. We have established the capability to perform the standard in vitro islet function assays (islet insulin release in low- and high-glucose media, viability assays, and more recently islet peri-fusion assays) and the in vivo NOD-scid transplant model. Due to the special expertise required for the latter, we have typically sent islets to collaborators at Vanderbilt University (Dr. Alvin Powers) for the NOD-scid assays but are working to establish the assay intramurally. We also performed new in vitro assays including microarray techniques, assays exploring the regulation of insulin biosynthesis at the translational level, and assays utilizing RT-PCR. Our collaborators are attempting to culture islets such that islet numbers increase in vitro. Our non-human primate experiments and have studied isolated monkey islets in vitro and have transplanted islets into diabetic non-human primate recipients. The non-human primate islet transplant model has yielded several important observations including: (1) NIH-isolated islets are viable and functional, (2) portal vein infusion is superior to intra-arterial infusion sites, (3) diabetes can be effectively and more safely induced (compared with the more typically employed surgical pancreatectomy approach) via intra-arterial infusion of a beta cell toxin call streptozotocin, (4) the immunohistological and metabolic consequences of islets transplanted into the liver, and (5) novel calcineurin phosphatase inhibitor free immunomodulatory regimens can support islet allograft survival. We have continued efforts to improve on the difficult and expensive primate model through collaborations with, for example, University of Maryland investigators who maintain a colony of spontaneously diabetic primates and with a Yale University interventional radiologist to find less invasive ways of transplanting the islets. All these studies are designed to support NIH islet transplant clinical trials. Under protocol, we transplanted allogeneic islets isolated from cadaveric donor pancreases into the portal veins of 6 patients with T1DM of at least 5 years duration, and with a clinical evidence of "brittle" disease. Of the six patients, four received a second islet infusion dose as per the protocol. All six patients are greater than a year post transplant and all patients have shown improvement in glycemia control, diminished requirements for exogenous insulin, and evidence of continued islet allograft function. While none of the patients had detectable endogenous insulin production prior to transplant as reflected by the absence of circulating C-peptide (even after attempts to stimulate C-peptide production using intravenous arginine), all patients display islet allograft function following transplant as reflected by insulin independence in 3 of the 6 enrollees (one returned recently to insulin therapy after 20 months insulin free- see below), decreased insulin requirements in all 6, and by improved Hgb A1c in all 6. Four of the 6 protocol enrollees tolerated the islet transplant procedure well. One patient suffered a partial portal vein thrombosis and requires chronic anticoagulation therapy, and another patient experienced a self limited intra-abdominal bleed following her second islet infusion, and required a 4 unit transfusion of irradiated packed red blood cells. One patient lost significant islet function over the past year but without evidence of immune rejection. Four of the 6 protocol enrollees managed the post transplant immunosuppressive regimen with tolerable toxicity. One patient elected to discontinue the regimen due to weight loss, fatigue, diarrhea, bone marrow suppression, and declining renal function. That patient had earlier displayed evidence suggesting she had rejected some of her transplanted islets, but she lost considerably more function once immunosuppressive agents were discontinued. Another patient, insulin independent for 20 months, recently developed a non-infectious pneumonitis associated with one of the immunosuppressive drugs, that drug was stopped, and her insulin requirements are now increasing. We performed detailed metabolic testing on the patients and found that even those insulin independent have a marginal islet mass and imperfect glycemia control. Insulin sensitivity, however, was normal. Due to our belief that the factors limiting islet transplantation (primarily the inadequate donor islet supply, and imperfect immunosuppressive regimens) were not being effectively and most safely addressed by the solitary islet transplant protocol, we suspended patient accrual. We are now preparing a combined kidney-islet transplant clinical protocol to test novel immunosuppressive regimens and islet transplantation techniques in patients with T1DM induced renal failure and therefore who already require a kidney allograft and chronic immunosuppression.